Caffeine (1,3,7-trimethylxanthine) and related methylxanthines are widely distributed in many plant species. Caffeine is also a major human dietary ingredient that can be found in common beverages and food products, such as coffee, tea, and chocolates. In pharmaceuticals, caffeine is used generally as a cardiac, neurological, and respiratory stimulant, as well as a diuretic (Dash et al., 2006). Hence, caffeine and related methylxanthines enter soil and water easily through decomposed plant materials and other means, such as effluents from coffee- and tea-processing facilities. Therefore, it is not surprising that microorganisms capable of degrading caffeine have been isolated from various natural environments, with or without enrichment procedures (Dash et al., 2006; Mazzafera, 2004). Bacteria use oxidative and N-demethylating pathways for catabolism of caffeine. Oxidation of caffeine by a Rhodococcus sp.-Klebsiella sp. mixed-culture consortium at the C-8 position to form 1,3,7-trimethyluric acid (TMU) has been reported (Madyastha and Sridnar, 1995). An 85-kDa, flavin-containing caffeine oxidase was purified from this consortium (Madyastha et al., 1996). Also, Mohapatra et al. (2006) purified a 65-kDa caffeine oxidase from Alcaligenes sp. Strain CF8. Cells of a caffeine-degrading Pseudomonas putida strain (ATCC 700097) isolated from domestic wastewater (Aganseitan, 2002) showed a fourfold increase in a cytochrome P450 absorption spectrum signal compared to cells grown on glucose. Recently, we reported a novel non-NAD(P)+-dependent heterotrimeric caffeine dehydrogenase from Pseudomonas sp. strain CBBI (Yu et al., 2008). This enzyme oxidized caffeine to TMU stoichiometrically and hydrolytically, without producing hydrogen peroxide. Further metabolism of TMU has not been elucidated.
Several caffeine-degrading bacteria metabolize caffeine via the N-demethylating pathway and produce theobromine (3,7-dimethylxanthine) or paraxanthine (1,7-dimethylxanthine) as the initial product. Theophylline (1,3-dimethylxanthine) has not been reported to be a metabolite in bacterial degradation of caffeine. Subsequent N-demethylation of theobromine or paraxanthine to xanthine is via 7-methyxanthine. Xanthine is further oxidized to uric acid by xanthine dehydrogenase/oxidase (Dash et al., 2006; Mazzafera, 2004). Although the identities of metabolites and the sequence of metabolite formation for caffeine N-demethylation are well established, there is very little information on the number and nature of N-demethylases involved in this pathway.